Molecular and Cell Biology of Cell Cycle Progression Revealed by Mammalian Cells Temperature-Sensitive in DNA Synthesis

1984 ◽  
pp. 229-248
Author(s):  
Rose Sheinin
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Temperature Sensitive ◽  
Cycle Progression

Effect of transfection manipulations on mouse cell cycle progression

1991 ◽  
Vol 69 (9) ◽  
pp. 665-669 ◽  
Author(s):  
Carol A. K. McBroom ◽  
Rose Sheinin
Keyword(s):  
Cell Cycle ◽  
Calcium Phosphate ◽  
Dna Synthesis ◽  
Key Words ◽  
Cell Cycle Progression ◽  
Mouse Cell ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Cellular Dna ◽  
Duplication Cycle

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphate-dimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak Sphase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1–S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.Key words: transfection, cell cycle progression.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

scholarly journals The Saccharomyces cerevisiae Anaphase-Promoting Complex Interacts with Multiple Histone-Modifying Enzymes To Regulate Cell Cycle Progression

2010 ◽  
Vol 9 (10) ◽  
pp. 1418-1431 ◽  
Author(s):  
Emma L. Turner ◽  
Mackenzie E. Malo ◽  
Marnie G. Pisclevich ◽  
Megan D. Dash ◽  
Gerald F. Davies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Chromatin Assembly ◽  
Temperature Sensitive ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Ubiquitin Ligase Complex ◽  
Genes Encoding ◽  
Dependent Cell ◽  
Histone Modifying Enzymes

ABSTRACT The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G1. Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56Ac) levels were as reduced as those of total H3, indicating that loading histones with H3K56Ac is unaffected in APC mutants. However, under restrictive conditions, H3K9Ac and dimethylated H3K79 (H3K79me2) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5 CA (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5 CA temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5 CA cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5 CA defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G1 and following G1 arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G1. To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.

Landomycin a inhibits DNA synthesis and cell cycle progression

1999 ◽  
Vol 9 (12) ◽  
pp. 1663-1666 ◽  
Author(s):  
Robert T Crow ◽  
Betty Rosenbaum ◽  
Roger Smith ◽  
Yu Guo ◽  
Kenneth S Ramos ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Landomycin A

scholarly journals Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

1999 ◽  
Vol 19 (7) ◽  
pp. 4623-4632 ◽  
Author(s):  
Masahiro Hitomi ◽  
Dennis W. Stacey
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Time Lapse ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

scholarly journals The second phase activation of protein kinase C δ at late G1 is required for DNA synthesis in serum-induced cell cycle progression

2003 ◽  
Vol 8 (4) ◽  
pp. 311-324 ◽  
Author(s):  
Koichi Kitamura ◽  
Keiko Mizuno ◽  
Akiko Etoh ◽  
Yoshiko Akita ◽  
Akitomo Miyamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Second Phase ◽  
Cycle Progression ◽  
Kinase C

scholarly journals Requirement for TAFII250 Acetyltransferase Activity in Cell Cycle Progression

2000 ◽  
Vol 20 (4) ◽  
pp. 1134-1139 ◽  
Author(s):  
Elizabeth L. Dunphy ◽  
Theron Johnson ◽  
Scott S. Auerbach ◽  
Edith H. Wang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Acetyltransferase Activity ◽  
Cycle Arrest ◽  
Protein Encoding ◽  
Acetyltransferase Domain ◽  
Mutant Cells

ABSTRACT The TATA-binding protein (TBP)-associated factor TAFII250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAFII250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G1. At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G1 cyclins, is dramatically reduced in the mutant cells. Here we demonstrate that the ability of the ts13 allele of TAFII250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5°C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAFII250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAFII150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or transcriptional defect observed at 39.5°C. These data suggest that TAFII250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes.

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